Toxicological Sciences

Published by Oxford University Press (OUP)
Online ISSN: 1096-0929
Publications
Article
Inhibitor-of-apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. Small-molecule IAP antagonists are currently being tested in clinical trials as novel cancer therapeutics. GDC-0152 is a small-molecule drug that triggers tumor cell apoptosis by selectively antagonizing IAPs. GDC-0152 induces NF-κB transcriptional activity leading to expression of several chemokines and cytokines, of which tumor necrosis factor alpha (TNF-α) is the most important for single-agent tumor activity. TNF-α is a pleiotropic cytokine that drives a variety of cellular responses, comprising inflammation, proliferation, and cell survival or death depending on the cellular context. As malignant and normal cells produce TNF-α upon IAP antagonism, increased TNF-α could drive both efficacy and toxicity. The toxicity profile of GDC-0152 in dogs and rats was characterized after intravenous dose administration once every two weeks for four doses. Findings in both species consisted of a dose-related, acute, systemic inflammatory response and hepatic injury. Laboratory findings included elevated plasma cytokines, an inflammatory leukogram, and increased liver transaminases with histopathological findings of inflammatory infiltrates and apoptosis/necrosis in multiple tissues; a toxicology profile consistent with TNF-α-mediated toxicity. Dogs exhibited more severe findings than rats, and humans did not exhibit these findings, at comparable exposures across species. Furthermore, elevations in blood neutrophil count, serum MCP-1 and other markers of inflammation corresponded to GDC-0152 exposure and toxicity and thus may have utility as safety biomarkers.
 
Article
The possible mechanism of the underlying nephropathy found in the rat toxicity study of FYX-051, a xanthine oxidoreductase inhibitor, was investigated. Rats received oral treatment of either 1 or 3 mg/kg of FYX-051, with and without citrate for four weeks to elucidate whether nephropathy could be caused by materials deposited in the kidney. Furthermore, analysis of the renal deposits in rats was also performed. Consequently, interstitial nephritis comprising interstitial inflammatory cell infiltration, dilatation, basophilia and epithelial necrosis of renal tubules and collecting ducts, deposits in renal tubules and collecting ducts, and so forth was seen in six of the eight rats and in all eight rats in the 1 and 3 mg/kg FYX-051 alone groups, respectively, with the intensity in the 3 mg/kg group being moderate to severe. In the simultaneous treatment with citrate group, however, no alterations were observed in the kidney, except for minimal interstitial nephritis in one instance in the 3 mg/kg FYX-051 + citrate group along with an increased urinary pH, leading to an increase in xanthine solubility. Analysis of intrarenal deposits showed that the entity would be composed of xanthine crystals. The present study, therefore, showed that nephropathy in rats occurring after the administration of FYX-051 was a secondary change caused by xanthine crystals being deposited in the kidney, and no other causes could be implicated in this kidney lesion.
 
The chemical structure of WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3-azidothymidine-5-(p-bromophenyl) methoxyalaninyl phosphate]. 
Blood Chemistry Profiles for B6C3F1 Mice Given WHI-07 Intravaginally for 13 Weeks 
Article
The zidovudine derivative, WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl) methoxyalaninyl phosphate), is a dual-function spermicidal agent with potent anti-HIV activity. In this study, groups of 20 female B6C3F1 mice were exposed intravaginally to a gel microemulsion containing 0, 0.5, 1.0, or 2.0% WHI-07, 5 days per week for 13 consecutive weeks. On a molar basis, these concentrations of WHI-07 are 1400- to 5700-times higher than its spermicidal EC(50) and 1.4 x 10(6) to 5.7 x 10(6) times higher than its in vitro anti-HIV IC(50). After 13 weeks of intravaginal treatment, mice were evaluated for toxicity. The endpoints that were used for evaluation included survival, body weight, hematologic and clinical chemistry profiles, absolute and relative organ weights, and histopathology. No effects related to WHI-07 treatments were observed on survival, mean body weight, and mean body-weight gain. Repeated intravaginal exposure of mice to WHI-07 for 13 weeks had no toxicologically significant effect on organ weights, and did not cause any adverse changes in hematology parameters or blood chemistry profiles. Extensive histopathologic examination of tissues showed no lesions of pathologic significance. Thus, intravaginal application of WHI-07, for up to 13 weeks, does not cause systemic toxicity.
 
The antiestrogenic effect of MeSO 2-PCBs on 17-estradiol (E2)-induced luciferase activity in human embryonic kidney (HEK) 293 cells stably transfected with a luciferase reporter gene construct and recombinant human estrogen receptor alpha (HEK293-ER-ERE.luc). The cells were treated with different concentrations of the individual MeSO 2-PCB congeners in the presence of either (A) 10 pM (approximate EC 50 ) of E2 or (B) 100 pM of E2. Luciferase activity was measured after 24 h of treatment as described under Materials and Methods. Luciferase activity is plotted as a percentage of the controls, i.e.. induction by E2 alone. Results for the E2 control and MeSO 2-PCB E2 treatments are expressed as means SD (3 sets, n 3 replicates). The significance of MeSO 2-PCB inhibition relative to the E2 controls is indicated (*p 0.05). 
Article
Methylsulfonyl (MeSO(2)) metabolites of polychlorinated biphenyls (PCBs) and 2,2-bis(4-chlorophenyl)-1,1-dichloroethene (4,4'-DDE), itself a metabolite of the insecticide 4,4'-DDT, are emerging as a major class of contaminants in the tissues of wildlife and humans. We investigated the antiestrogenic capacity and potencies of 3'- and 4'-MeSO(2)-2,2',4,5,5'-pentachlorobiphenyl (CB101) and -2,2',4,5'-tetrachlorobiphenyl (CB49), which are among the most environmentally persistent MeSO(2)-PCBs, and 3-MeSO(2)-4,4'-DDE on estrogen receptor (ER)-dependent gene expression in four cell-based bioassay systems. Congener- and concentration-dependent antagonism of 17beta-estradiol (E2)-induced gene expression, rather than induction of ER-dependent gene expression, was observed for the MeSO(2)-PCBs on lucifierase activity in stably transfected human breast adenocarcinoma T47D cells (ER-CALUX) and vitellogenin (vtg) production in primary hepatocytes from male carp fish (Cyprinus carpio) (CARP-HEP/vtg). 4'-MeSO(2)-CB101 and -CB49 had the highest antagonistic potency (i.e., maximum inhibition of about 70%, LOECs of 1.0 microM and 2.5 microM), whereas 3'-MeSO(2)-CB101 and -CB49 were less antagonistic; the precursor CB101 and MeSO(2)-PCB analog MeSO(2)-2,5-dichlorobenzene had no effect. Relative to the 4-MeSO(2)-PCBs, tamoxifen (IC(50), 0.06 microM and 0.7 microM) was about 40 and 7 times more potent in the ER-CALUX and CARP-HEP/vtg assays, respectively. Congener- and concentration-dependent effects on aryl hydrocarbon receptor-mediated induction of EROD activity (carp hepatocytes), luciferase expression (H4IIE rat hepatoma [H4IIE.luc] cell line), or cell viability were not observed. 3-MeSO(2)-4,4'-DDE was neither estrogenic nor antiestrogenic in either of the bioassays. Inhibitory trends for the MeSO(2)-PCBs in a bioassay based on stably transfected human embryonic kidney cell (HEK293-ERalpha-ERE) were similar to the ER-CALUX and CARP-HEP/vtg bioassays, whereas the antagonism was weaker in a related HEK293-ERbeta-ERE bioassay. Our findings suggest that the 4'-MeSO(2)-PCBs are antiestrogenic in vitro via a reversible or surmountable interaction with fish or human ER, and that the interaction with human ERalpha is apparently favored over ERbeta. MeSO(2)-PCB metabolites are persistent and bioaccumulative contaminants, and therefore, could be potentially active as environmental antiestrogens in wildlife and humans.
 
Article
1,1-Dichloroethane (DCE) is a solvent that is often found as a contaminant of drinking water and a pollutant at hazardous waste sites. Information on its short- and long-term toxicity is so limited that the U.S. EPA and ATSDR have not established oral reference doses or minimal risk levels for the volatile organic chemical (VOC). The acute oral LD(50) in male Sprague-Dawley (S-D) rats was estimated in the present study to be 8.2 g/kg of body weight (bw). Deaths appeared to be due to CNS depression and respiratory failure. In an acute/subacute experiment, male S-D rats were given 0, 1, 2, 4, or 8 g DCE/kg in corn oil by gavage for 1, 5, or 10 consecutive days. The animals were housed in metabolism cages for collection of urine and sacrificed for blood and tissue sampling 24 h after their last dose. There were decreases in body weight gain and relative liver weight at all dosage levels, as well as increased renal nonprotein sulfhydryl levels at 2 and 4 g/kg after 5 and 10 days. Elevated serum enzyme levels, histopathological changes, and abnormal urinalyses were not manifest. For the subchronic study, adult male S-D rats were gavaged with 0.5, 1, 2, or 4 g DCE/kg 5 times weekly for up to 13 weeks. Animals receiving 4 g/kg exhibited pronounced CNS depression, with more than one-half dying by week 11. The 2-g/kg rats exhibited moderate CNS depression. One 2-g/kg rat died during week 6. There were very few manifestations of organ damage in animals that succumbed or in survivors at any dosage level. Decreases in bw gain and transient increases in enzymuria were noted at 2 and 4 g/kg. Serum enzyme levels and blood urea nitrogen were not elevated, nor were glycosuria or proteinuria present. Chemically induced histological changes were not seen in the liver, kidney, lung, brain, adrenal, spleen, stomach, epididymis, or testis. Hepatic microsomal cytochrome P450 experiments revealed that single, high oral doses of DCE did not alter total P450 levels, but did induce CYP2E1 levels and activity and inhibit CYP1A1 activity. These effects were reversible and regressed with repeated DCE exposure. There was no apparent progression of organ damage during the 13-week subchronic study, nor appearance of adverse effects not seen in the short-term exposures. One g/kg orally (po) was found to be the acute, subacute, and subchronic LOAEL for DCE, under the conditions of this investigation. In each instance, 0.5 g/kg was the NOAEL.
 
Cumulative mass of TCEOH (upper) and TCA (lower) excreted over 70 h postexposure for exposures with and without prior ethanol consumption. Data represent mean interpolated values at specified times from all 10 subjects (error bars ¼ 1 SD).
Percent increase in apparent metabolic clearance of 1,1,1trichloroethane with prior ethanol consumption for each subject. Mean increase shown (bold line) along with 95% CIs for the mean (dashed line).
Article
This study evaluated the effect of moderate doses of ethanol over a short period of time on the toxicokinetics of an organic solvent, 1,1,1-trichloroethane. A group of 10 moderate drinkers were recruited and exposed via inhalation for 2 h to a low concentration of 1,1,1-trichloroethane (175 ppm) on two separate occasions. Subjects were administered ethanol (0.35 g/kg body weight) on each of the 7 days preceding one of the exposures. Blood and urine samples were collected during and following each exposure, with blood analyzed for 1,1,1-trichloroethane and urine analyzed for the metabolites of 1,1,1-trichloroethane: trichloroethanol and trichloroacetic acid. Prior ethanol consumption resulted in a significant increase in apparent metabolic clearance of 1,1,1-trichloroethane (mean increase = 25.4%). The results of this study demonstrate that ethanol consumption over time can affect the rate at which an organic solvent is cleared through metabolism in humans. For chemicals with toxic metabolic products, this inductive effect of ethanol consumption on the rate of biotransformation could be potentially harmful to exposed individuals. Metabolic clearance of compounds with high hepatic extraction may not be affected by enzyme induction as it is likely that these compounds are essentially completely metabolized while passing through the liver.
 
Article
1,1,1-Trichloroethane (TRI) is a widely used solvent that has become a frequent contaminant of drinking water supplies in the U.S. There is very little information available on the potential for oral TRI to damage the liver or to alter its P450 metabolic capacity. Thus, a major objective of this investigation was to assess the acute, short-term, and subchronic hepatotoxicity of oral TRI. In the acute study, male Sprague-Dawley (S-D) rats were gavaged with 0, 0.5, 1, 2, or 4 g TRI/kg bw and killed 24 h later. No acute effects were apparent other than CNS depression. Other male S-D rats received 0, 0.5, 5, or 10 g TRI/kg po once daily for 5 consecutive days, rested for 2 days, and were dosed for 4 additional days. Groups of the animals were sacrificed for evaluation of hepatotoxicity 1, 5, and 12 days after initiation of the short-term experiment. This dosage regimen caused numerous fatalities at 5 and 10 g/kg, but no increases in serum enzymes or histopathological changes in the liver. For the subchronic study, male S-D rats were gavaged 5 times weekly with 0, 0.5, 2.5, or 5.0 g TRI/kg for 50 days. The 0 and 0.5 g/kg groups were dosed for 13 weeks. A substantial number of rats receiving 2.5 and 5.0 g/kg died, apparently due to effects of repeated, protracted CNS depression. There was evidence of slight hepatocytotoxicity at 10 g/kg, but no progression of injury nor appearance of adverse effects were seen during acute or short-term exposure. Ingestion of 0.5 g/kg over 13 weeks did not cause apparent CNS depression, body or organ weight changes, clinical chemistry abnormalities, histopathological changes in the liver, or fatalities. Additional experiments did reveal that 0.5 g/kg and higher doses induced hepatic microsomal cytochrome P450IIE1 (CYP2E1) in a dose- and time-dependent manner. Induction of CYP2E1 activity occurred sooner, but was of shorter duration than CYP2B1/2 induction. CYP1A1 activity was not enhanced. In summary, 0.5 g/kg po was the acute, short-term, and subchronic NOAEL for TRI, for effects other than transient CYP2E1 induction, under the conditions of this investigation. Oral TRI appears to have very limited capacity to induce P450s or to cause liver injury in male S-D rats, even when administered repeatedly by gavage in near-lethal or lethal dosages under conditions intended to maximize hepatic effects.
 
Locomotor activity during exposure to a subset of nine TRI concentrations, relative to control activity. Locomotor activity was recorded in 1-min bins. Each data point represents the mean of 5 mice. The boundary zone encompassing the 95% confidence interval ( 2, SD) for mean control activity is represented by the shaded region.
Scatter plot relating blood and brain concentrations of TRI as measured at comparable times and exposure levels. Each data point (circle) represents the mean blood and brain concentration of 2 (10,000 and 14,000 ppm) or 4 (500, 1000, 2000, 4000, 6000, 8000, and 12,000 ppm) mice after 6, 12, 18, 24, or 30 min of exposure to one of nine TRI concentrations. The equation of the 1st order regression line: y 0.954x 5.095.  
Scatter plots relating locomotor activity and (a) blood and (b) brain concentrations of TRI. Each data point represents the mean blood or brain concentration of 2 (10,000 and 14,000 ppm) or 4 (500, 1000, 2000, 4000, 6000, 8000, and 12,000 ppm) mice after 6, 12, 18, 24, or 30 min of exposure to 500 (circle), 1000 (square), 2000 (1 triangle), 4000 (2 triangle), 6000 (diamond), 8000 (hexagon), 10,000 (circle ), 12,000 (square ) or 14,000 ppm TRI (triangle ), as well as the mean locomotor activity (relative to air control) of 5 mice at comparable times and equivalent exposure levels. The equations and correlation coefficients for the 2nd order regression lines are y – 6.755 0.485x – 0.0017x 2 (blood; r 0.81); y – 4.443 0.412x – 0.0014x 2 (brain; r 0.80).  
Scatter plots relating locomotor activity and internal measures of TRI exposure. Each data point represents an internal measure of TRI exposure (blood C max , brain C max , blood AUC, brain AUC), as well as the total locomotor activity count (relative to air control) during 30-min exposures to 500 ppm (white circle, black circle), 1000 ppm (white square, black square), 2000 ppm (white 1 triangle, black 1 triangle), 4000 ppm (white 2 triangle, black 2 triangle), 6000 ppm (white diamond, black diamond), 8000 ppm (white hexagon, black hexagon), 10,000 ppm (white circle , black circle ), 12,000 ppm (white square , black square ) or 14,000 ppm TRI (white triange , black triangle ). C max values are the maximum blood and brain concentrations of TRI achieved during 30-min exposures; AUC values are the areas under the blood and brain concentration vs. time curves from 0 to 30 min. The equations and correlation coefficients for the 2nd order regression lines are: y –188.323 11.063x – 0.0333x 2 (blood C max , r 0.94); y –131.496 9.197x – 0.0247x 2 (brain C max , r 0.95); y –176.204 0.455x – 0.0000565x 2 (blood AUC, r 0.95); y –157.226 0.439x – 0.0000558x 2 (brain AUC, r 0.93).  
Article
Despite the central nervous system (CNS) being a target of virtually all solvents, few solvents have been thoroughly studied for their effects on unlearned animal behaviors. Of the solvents that have been studied, little is known about the relationship of exposure concentration to behavioral effect, and quantitative data relating the toxicologically important target organ (i.e., brain) dose to behavioral effect are almost non-existent. To examine the concentration- and time-dependency of effects of 1,1, 1-trichloroethane (TRI) on behavior, male albino Swiss-Webster mice were exposed to TRI (500-14,000 ppm) in static inhalation chambers for 30 min, during which locomotor activity was measured. Separate mice were exposed to the same concentrations under identical conditions for 6, 12, 18, 24, and 30 min, to determine blood and brain concentrations versus time profiles for TRI. This allowed for the relationships between blood and brain concentrations of TRI and locomotor activity to be discerned. The lowest TRI concentrations studied (500-2000 ppm) had no statistically significant effect on activity, intermediate concentrations (4000-8000 ppm) increased activity immediately to levels that remained constant over time, and higher concentrations (10,000-14,000 ppm) produced biphasic effects, i.e., increases in activity followed by decreases. 1,1, 1-Trichloroethane concentrations in blood and brain approached steady-state equilibria very rapidly, demonstrated linear kinetics, and increased in direct proportion to one another. Locomotor activity increased monophasically ( approximately 3.5-fold) as solvent concentrations increased from approximately 50-150 microg/g brain and microg/ml blood. As concentrations exceeded the upper limit of this range, the activity level declined and eventually fell below the control activity level at approximately 250 microg/g brain and microg/ml blood. Regression analyses indicated that blood and brain concentrations during exposure were strongly correlated with locomotor activity, as were measures of internal dose integrated over time. The broad exposure range employed demonstrated that TRI, like some classical CNS depressants, is capable of producing biphasic effects on behavior, supporting the hypothesis that selected solvents are members of the general class of CNS depressant drugs. By relating internal dose measures of TRI to locomotor activity, our understanding of the effects observed and their predictive value may be enhanced.
 
Article
The aim of this study was to determine the toxicokinetics of inhaled 1,1,1,3,3-pentafluoropropane (HFC-245fa) in humans. Five healthy volunteers of each sex were exposed in random order to 0, 100, or 300 ppm HFC-245fa for 2 h at light exercise (50 W) in an exposure chamber. Capillary blood, urine, and exhaled air were sampled up to 22 h postexposure and analyzed for HFC-245fa. In addition, the metabolites fluoride, 3,3,3-trifluoropropionic acid (TFPA), and trifluoroacetic acid (TFAA) were analyzed in urine. Symptoms of irritation and central nervous system effects were rated in visual analogue scales. Various biochemical (aspartate-amino transferase, alanine-amino transferase, alkaline phosphate, glutamyl transferase, urate, creatine kinase [CK], and CK muscle brain) and inflammatory markers (serum amyloid A protein, fibrinogen, D-dimer, C-reactive protein, and interleukin-6) in plasma were analyzed. The initial increase in blood was fast and an apparent steady state was reached within a few minutes at both exposure levels. The postexposure decrease in blood was equally fast and parallel to that in exhaled air. Only minor amounts of unchanged HFC-245fa were excreted in breath (0.7% of inhaled) and urine (0.001%). The observed time courses in blood and breath agreed reasonably well those obtained by physiologically based pharmacokinetic (PBPK) modeling. The PBPK simulations indicate a relative uptake during exposure of 2.1%. TFPA was not detected in urine, and no increase in TFAA or fluoride above background was seen, suggesting little or no metabolism, the calculated minimum detectable metabolism being 0.001% of the inhaled amount. The symptom ratings revealed no HFC-245fa-related effects. None of the biochemical markers was affected. The changes in inflammatory markers, some of which are statistically significant, were not consistent with an inflammatory response.
 
Article
1,1,1,3,3-Pentafluoropropane (HFC 245fa) is a volatile, low boiling liquid. It was inactive in a reverse mutation (Ames) assay using five strains of Salmonella typhimurium and one strain of Escherichia coli. It was also inactive in an in vivo mouse micronucleus assay with exposures of 101,000 ppm. In a chromosome aberration study with human lymphocytes, some activity was seen when cell cultures were exposed to atmospheres of 30% v/v or higher for 24 h without metabolic activation. No activity was seen in assays using less than 30% v/v or exposure times of less than 24 h. No activity was seen in the presence of metabolic activation even with exposures of 70%. It was not toxic by the dermal route. There was no mortality or significant signs of toxicity when rats and mice were given 4- h exposures to levels of 203,000 ppm or 101,000 ppm of HFC 245fa, respectively. In a cardiac sensitization study with dogs involving intravenous administration of epinephrine, the no observed effect level (NOEL) was 34,000 ppm and the threshold for a response was 44,000 ppm. In a rat inhalation, developmental toxicity study, a slight reduction in pup weight was seen at 50,000 ppm, but not at 10,000 ppm. There were no developmental effects at any level. A series of three inhalation toxicity studies were conducted. All involved daily 6-h exposures up to 50,000 ppm. The first study involved 14 consecutive snout-only exposures. There were no treatment-related effects on body weight, survival, or histologic parameters. BUN, GPT, and GOT levels frequently were elevated compared to controls , whereas cholesterol levels tended to be lower. The second study involved 28 consecutive whole-body exposures. Again, there were no treatment related effects on body weight, survival, or histological parameters. Urine volume was increased. Increases were also seen in several red blood cell parameters. These may be related to partial dehydration. Increases were seen in BUN levels and alkaline phosphatase (AP), GPT, GOT and CPK activities, primarily in rats exposed at 10,000 and 50,000 ppm. Urinary fluoride levels were also elevated in an exposure- related pattern. In the third study, whole-body exposures were conducted 5 days per week for 13 weeks. There were no treatment-related effects on survival, clinical observations, body weight gain, or food consumption. Urine volumes were increased, urinary fluoride levels were elevated, and increases were seen in red blood cell counts, and related parameters and increases were seen in AP, GOT, GPT and CPK activities. These effects were seen in the 10,000 and 50,000 ppm exposure level groups. Histopathologic examination did not show any effects on the kidney, liver, or lungs. There was an increased incidence of myocarditis in all animals exposed at 50,000 ppm and the majority exposed at 10,000 ppm. It was described as mild. Based on these findings, 2000 ppm appears to be a no observed adverse effect level.
 
Reproductive Outcome of Rats Exposed by Inhalation to HCC-230fa 
Incidence of Fetal Variations 
Article
The potential developmental toxicity and the in vitro and in vivo genotoxicity of HCC-230fa were assessed. In the developmental toxicity study, groups of 25 mated Crl:CD(R)(SD)BR rats were exposed (whole body) by inhalation to HCC-230fa over days 7-21 of gestation; the day of confirmed mating was designated as gestation day 1 (GD1). Exposures were 6 h per day at concentrations of 0, 0.5, 2.5, or 25 ppm. Body weight, food consumption, and clinical observation data were collected during the study. On day 22 of gestation, the dams were euthanized and examined grossly. The fetuses were removed and subsequently weighed, sexed, and examined for external, visceral, head, and skeletal alterations. Evidence of maternal and developmental toxicity was observed at 25 ppm and was noted as significant, compound-related reductions in mean maternal body weight, weight change, and food consumption. Significant fetal effects also were observed at 25 ppm as compound-related reductions in mean fetal weight and increased fetal malformations (filamentous tail, situs inversus, absent vertebrae) and variations (rudimentary cervical ribs, delayed sternebral ossification). There was no evidence of either maternal or developmental toxicity at 0.5 or 2.5 ppm. The genotoxicity of HCC-230fa was examined in a bacterial reversion assay and in erythrocyte micronucleus studies in two species by different routes of administration. No increases in the number of revertants were observed in the bacterial reversion assay. In one micronucleus study, HCC-230fa was administered by inhalation to rats as part of a 90-day study at doses indicated above. For the second study, ICR mice were given a single ip dose at 0, 166, 330, or 660 mg/kg. In both micronucleus studies, a significant increase in micronucleated erythrocytes was observed. The results of these studies suggest that HCC-230fa affects rapidly dividing cells and may have long-term consequences for occupational exposures.
 
White Blood Cell and Lymphocyte Counts for Male and Female Rats 
Mean body weights of male rats exposed for 90 days to HCC-230fa concentrations of 0, 0.5, 2.5, or 25 ppm. Standard deviations on the mean values were approximately 10% or less. *Indicates animals fasted prior to necropsy. 
Mean body weights of female rats exposed for 90 days to HCC230fa concentrations of 0, 0.5, 2.5, or 25 ppm. Standard deviations on the mean values were approximately 10% or less. *Indicates animals fasted prior to necropsy. 
Sperm Analysis in Male Rats At 0-Day Recovery 
Sperm Analysis in Male Rats At 1-Month Recovery 
Article
Male and female rats were exposed by inhalation (whole body) to HCC-230fa (1,1,1,3,3,3-hexachloropropane) for 6 h/day, 5 days/week over a 15-week period. Concentrations of 0, 0.50, 2.5, and 25 ppm were studied. A total of eight groups/sex were exposed. Four groups of male and four groups of female rats were used to measure clinical signs and growth, clinical pathology, and tissue pathology. The remaining four groups of male rats were used for immunotoxicological and sperm assessment evaluations, and the remaining four groups of female rats were used for immunotoxicological evaluation. Following the exposure period, surviving male rats were kept for a 1- or 3-month recovery period. Male and female rats exposed to 25 ppm had lower mean body weights, mean body weight gains, and food consumption during the exposure period. Male and female rats exposed to 25 ppm and sacrificed immediately after the exposure period had minimally decreased total leukocyte and lymphocyte counts. These changes were considered to be marginally adverse. Pathologic examination revealed hepatocellular hypertrophy in 0-day recovery males and an increased incidence and/or severity in chronic progressive nephropathy in 0-day, 1-month recovery, and 3-month recovery males at 25 ppm. No other pathological changes, including the testis, epididymis, and other accessory sex organs, were noted in rats during the study. Evaluation of sperm parameters at the end of the exposure period showed statistically significant decreases in epididymal sperm number per cauda epididymis, percent motile sperm, and percent normal sperm morphology at 25 ppm. The biological significance of the slight changes observed in the sperm parameters in the absence of histopathological changes is unclear. After a 1-month recovery period, no biologically significant differences in sperm parameters were noted at 25 ppm compared with controls. Exposure to HCC-230fa did not significantly alter the primary humoral immune response to sheep red blood cell (SRBC). Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) was considered to be 2.5 ppm.
 
Article
Groups of 25 female F344 rats and 25 female B6C3F1 mice were exposed to 0, 30, 300, 600, or 1200 ppm tetrafluoroethylene (TFE) by inhalation for up to 12 days. Another set of 25 female rats and 25 female mice of the same strains were given 0, 5, 20, or 50 mg/kg of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS) by oral gavage for 12 days. Both 12-day exposure regimens consisted of exposures for 5 consecutive days, a weekend with no exposures, and 4 consecutive daily exposures following the weekend. Five animals per group were sacrificed after the first exposure, the fifth exposure, and the ninth exposure for evaluation of cell proliferation in the liver and kidney. The remaining animals in each group (up to 10) were sacrificed after the ninth exposure (test day 12) for pathological evaluation of the liver, kidney, and spleen. Clinical pathology evaluations were performed on test day 11 or 12. Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice, but no histopathological changes in the liver. In the kidney, administration of TFE-CYS by gavage caused severe microscopic changes in rats, moderate-to-severe changes in mice, and no microscopic changes in the liver. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused increased liver weights and cell proliferation in the liver of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most pronounced after 5 days of exposure, and less evident or absent after 12 days of exposure. In the kidney, the cell proliferation and histopathologic response in rats was generally more pronounced than in mice. Kidney damage and cell proliferation were confined to the pars recta (P3) of the outer stripe of the outer medulla and medullary rays. Tubules in mice exposed to TFE and TFE-CYS had mostly regenerating cells by test day 12, while in rats the tubules still showed marked degeneration along with regeneration by the end of the study. The cortical labyrinth (P1 and P2 segments) was also affected at the 50 mg/kg dose of TFE-CYS in rats. Rats exposed to 50 mg/kg TFE-CYS had a mild anemia, and rats exposed to 1200 ppm TFE had slight, biologically inconsequential decreases in erythrocyte mass that may have been compound-related. In spite of the rather pronounced histopathologic changes in the kidneys of rats exposed to TFE-CYS, there was no clinical chemistry evidence for decreased kidney function. Increased levels of urinary fluoride were present in rats exposed to 300 ppm and greater of TFE, and in rats exposed to 20 and 50 mg/kg TFE-CYS. The spleen was not affected in this study. Overall, the results of this study suggest that effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-week exposure, as all effects that were seen with TFE were also seen with TFE-CYS.
 
BD metabolism and formation of N-terminal valine adducts. The numbers in EB-diol are based on the metabolism and are not in accordance with IUPAC nomenclature.
Ion chromatograms of pyr-Val from a B6C3F1 mouse (A) and a F344 rat (B) exposed to 1 ppm BD for 10 days. Shown are the ion transitions of the doubly charged analyte (m/z 417.3-158.3, top) and internal standard peptides (m/z 418.8-158.7, bottom).
Exposure-response of pyr-Val in male (solid lines) and female (dashed lines) B6C3F1 mice and F344 rats. (A) Mice were exposed to BD for 10 days. (B) Mice were exposed to BD for 20 days. (C) Rats were exposed to BD for 10 days. (D) Rats were exposed to BD for 20 days. Rodents were exposed to BD by inhalation, and globin was obtained and analyzed for pyr-Val as described in the ''Material and Methods'' section. Inserts in (A) and (C) show exposure-responses at exposures below 7.5 ppm BD.
Article
1,3-Butadiene (BD) is a known rodent and human carcinogen that is metabolized mainly by P450 2E1 to three epoxides, 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol. The individual epoxides vary up to 200-fold in their mutagenic potency, with DEB being the most mutagenic metabolite. It is important to understand the internal formation of the individual epoxides to assign the relative risk for each metabolite and to understand the molecular mechanisms responsible for extensive species differences in carcinogenicity. This study presents a comprehensive exposure-response for the formation of the DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)-valine (pyr-Val) in mice and rats. Using nano-ultra high pressure liquid chromatography-tandem-mass spectrometry allowed analysis of pyr-Val in mice and rats exposed to BD as low as 0.1 and 0.5 ppm BD, respectively, and demonstrated significant differences in the amounts and exposure-response of pyr-Val formation. Mice formed 10- to 60-fold more pyr-Val compared to rats at similar exposures. The formation of pyr-Val increased with exposures, and the formation was most efficient with regard to formation per parts per million BD at low exposures. While formation at higher exposures appeared linear in mice, in rats formation saturated at exposures ≥ 200 ppm for 10 days. In rats, amounts of pyr-Val were lower after 20 days than after 10 days of exposure, suggesting that the lifespan of rat erythrocytes may be shortened following exposure to BD. This research supports the hypothesis that the lower susceptibility of rats to BD-induced carcinogenesis results from greatly reduced formation of DEB following exposure to BD. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] /* */
 
Article
S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), a model nephrotoxicant in mice, causes acute tubular necrosis and death at high doses. Our earlier studies revealed that renal tissue repair was critical for survival in mice with DCVC nephrotoxicity. The objective of this study was to investigate if increasing renal tissue repair could protect mice from the lethal outcome of DCVC. Male Swiss Webster (SW) mice were administered a low dose of DCVC (15 mg/kg, ip) 72 h before injection of a normally lethal dose of DCVC (75 mg/kg, ip); this resulted in 100% protection against the lethal effect of DCVC. Because DCVC caused approximately two fold decrease in cytosolic and mitochondrial beta-lyase activity, the possibility that DCVC protection may be caused by decreased bioactivation was examined. Mercuric chloride (HgCl2, 6 mg/kg), a nephrotoxicant with no effect on beta-lyase activity, was administered 96 h before a lethal dose of DCVC. This also resulted in 100% protection from the lethal effect of DCVC. In both studies total glutathione was unchanged at any time after the lethal dose of DCVC was administered, obviating the role of glutathione in protection. In both cases the augmented and sustained tissue repair induced by priming dose and documented by 3H-thymidine pulse labeling and immunocytochemistry for proliferating cell nuclear antigen resulted in 100% survival in spite of the extensive renal injury. These findings suggest that stimulation of renal tubular repair by the priming dose, through augmented cell division, and the resistance of new cells to mechanisms of progression of injury, underlies auto- and heteroprotection against DCVC. The molecular mechanisms may have potential application in pharmacotherapeutic intervention for treatment of acute renal failure.
 
Article
3-Butene-1,2-diol (BDD) is a major metabolite of 1,3-butadiene (BD), but the role of BDD in BD toxicity and carcinogenicity remains unclear. In this study, the acute toxicity of BDD was investigated in male Sprague-Dawley rats and B6C3F1 mice. Of the rats given 250 mg/kg BDD, 2 out of 4 died within 24 h; rats experienced hypoglycemia, significant alterations of liver integrity tests, and had lesions in the liver 4 h after treatment, but no lesions were detected in extrahepatic tissues. Rat hepatic GSH and GSSG levels were significantly depleted at both 1 and 4 h after the BDD treatment. Rats administered 200 mg/kg BDD also had liver lesions but no death or hypoglycemia was observed four or 24 h after treatment; these rats had depleted hepatic GSH and GSSG levels at 1 h but not at 4 or 24 h after treatment. Mice administered 250 mg/kg BDD exhibited modest alterations of liver integrity tests, but no death, hypoglycemia, or lesions in any tissue, and hepatic GSH and GSSG levels were depleted at 1 h but not at 4 h. The plasma half-life of BDD was four times longer in rats than in mice. Additional studies in rats showed the depletion of hepatic GSH and GSSG preceded the BDD-induced hypoglycemia and hepatotoxicity. Thus, the long half-life of BDD in rat plasma and the sustained depletion of hepatic GSH and GSSG may in part explain the higher sensitivity of the rat to BDD-induced hepatotoxicity. Furthermore, the results indicate that BDD may play a role in BD-induced toxicity.
 
Cadmium content of organs of mouse dams at various stages of gestation or lactation. Values are mean SE. Closed squares give results from MTN dams; open squares from MTN NP20 controls (20 days of 109 Cd exposure, nonpregnant mice, equivalent to LD 1). Closed circles give results from MT1,2KO dams; open circles from MT1,2KO NP20 controls. GD 7, GD 14, GD 17, NP20, and LD 11 MTN values are means from six, four, four, six, and eight mice, respectively, with exception of LD 11 blood (n 7) and NP20 liver (n 5). Corresponding MT1,2KO group sizes are three, six, six, six, and five mice, respectively, with the exception of LD 11 blood (n 4). Cadmium exposures and abbreviations as in Table 1. *Indicates MT1,2KO value significantly different from corresponding MTN value (p 0.05, Student's t-test, using the assumption of unequal variances).  
Article
Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of (109)CdCl(2) (0.15 ng Cd/ml; 0.074 micro Ci (109)Cd/ml). (109)Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of (109)Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific (109)Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental (109)Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal (109)Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of (109)Cd transferred to pups via milk; furthermore, 85-90% of total pup (109)Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal (109)Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.
 
Article
1,2-dichloropropane (1,2-DCP) has recently been reclassified from not classifiable as to its carcinogenicity to humans (Group 3) to carcinogenic to humans (Group 1) by the International Agency for Research on Cancer. This was based on the findings of epidemiological studies in Japan that occupational exposure to paint stripers containing 1,2-DCP was associated with increased cholangiocarcinomas. It is known that 1,2-DCP is negative for cholangiocarcinogenicity in rats and mice. However, its toxicity and carcinogenicity has not been examined in hamsters and little is known about the regulation of its metabolism in hamsters. The purpose of the present study was to determine the hepatobiliary toxicity of 1,2-DCP in hamsters and to characterize and compare the altered patterns of hepatic xenometabolic enzymes in hamsters and mice. Male Syrian hamsters and male B6C3F1 mice were treated with various doses of 1,2-DCP for 4 hours or 3 days or 4 weeks. These experiments demonstrated that a high dose of 1,2-DCP induced centrilobular hepatocellular necrosis in hamsters. CYP2E1 is possibly the key enzyme responsible for bioactivation and the consequent hepatocytotoxicity of 1,2-DCP, and GSH conjugation catalyzed by GST-T1 may exert a cytoprotective role in hamsters and mice. Notably, the expression pattern of GST-T1 in bile duct epithelial cells differed between hamsters and mice: GST-T1 was expressed in bile duct epithelial cells of mice but not hamsters. This indicates that responses to 1,2-DCP in the bile duct of hamsters might differ from that of mice, and suggests that long-term studies are necessary to clarify the chalangiocarcinogenicity of 1,2-DCP in hamsters, though no biliary toxicity was observed in the present short-term experiments. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.
 
Article
Naphthalene is an important industrial chemical, which has recently been shown to cause tumors of the respiratory tract in rodents. It is thought that one or more reactive metabolites of naphthalene, namely, naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) contribute to the tumorigenicity of this chemical. These electrophiles are all capable of covalent binding to macromolecules including DNA and proteins. The stability of cysteinyl adducts of NPO, 1,2-NPQ, and 1,4-NPQ were investigated in both hemoglobin (Hb) and albumin (Alb) of male F344 rats following a single administration of 2 different doses (400 or 800 mg naphthalene per kg body weight). To assess the stability of Alb adducts, we compared the rates of NPO-Alb turnover (half-life of approximately 2 days) and 1,2-NPQ-Alb (half-life of approximately 1 day) to the normal turnover rate of Alb in the rat (half-life = 2.5-3 days). Based on the rapid turnover of these adducts relative to Alb itself, we concluded that they were unstable. However, the stability of Alb adducts was not affected by the dose of naphthalene administered (400 or 800 mg/kg). In contrast, NPO-Hb adducts were relatively stable (rate constant of adduct instability <or= 0.01) following a 400 mg/kg dose of naphthalene, but their stability could not be estimated following an 800 mg/kg dose due to high variability among animals. The rate constants of adduct instability obtained in this study allow application of NPO and NPQ adducts to estimate the exposure to reactive electrophilic metabolites of naphthalene in the rat. In addition, some limitations of current methods for estimating adduct instability were identified.
 
Article
1,2-Dichlorobenzene (1,2-DCB) is a potent hepatotoxicant in male Fischer 344 (F344) rats and previous studies have suggested that reactive oxygen species may play a role in the development of hepatotoxicity. Since reactive oxygen species can damage lipid membranes, this study was conducted to determine the extent of lipid peroxidation after administration of 1,2-DCB by immuno-histochemical analysis of 4-hydroxynonenal (4-HNE) protein adduct formation in liver and conjugated diene formation in liver and serum. The contribution of Kupffer cells to the lipid peroxidation was also investigated. Male F344 rats were administered 1,2-DCB (3.6 mmol/kg i.p. in corn oil) and killed at selected times between 3 and 48 h. Time course studies revealed the greatest abundance of 4-HNE protein adducts in the centrilobular regions of the liver 24 h after 1,2-DCB administration, with much lower levels at 16 h. Adducts were present in necrotic and vacuolized centrilobular hepatocytes of 1,2-DCB treated rats but not in livers of controls. Further, conjugated dienes were significantly increased in liver and serum 16 and 24 h after 1,2-DCB administration, peaking at 24 h. These data correlated with hepatocellular injury, determined by serum alanine aminotransferase activity and histopathological evaluation, which was markedly elevated within 16 h and peaked at 24 h. When rats were pretreated with gadolinium chloride (GdCl3; 10 mg/kg i.v. 24 h prior to 1,2-DCB), an inhibitor of Kupffer cells, hepatotoxicity was decreased by 89 and 86%, at 16 and 24 h, respectively. Conjugated diene concentrations were decreased to control values at these times after 1,2-DCB administration. Moreover, no 4-HNE protein adducts were detected in livers of 1,2-DCB-treated rats pretreated with GdCl3. Finally, Kupffer cells isolated from 1,2-DCB-treated rats produced significantly more superoxide anion than Kupffer cells isolated from vehicle controls. These data, along with previous findings, suggest that lipid peroxidation associated with 1,2-DCB is mediated in part by Kupffer cell-derived reactive oxygen species.
 
Article
1,2-dichlorobenzene (1,2-DCB), an industrial solvent, is a known hepatotoxicant. Two oxidative events in the liver contribute to 1,2-DCB-induced liver injury: an initial hepatocellular oxidative stress, followed by oxidant stress associated with an inflammatory response. We hypothesize that the initial hepatocellular oxidative event triggers molecular and cellular processes within hepatocytes that lead to the production of factors that contribute to Kupffer cell (KC) activation and upregulation of the inflammatory cascade. To investigate the molecular effects of 1,2-DCB, primary cultures of Fischer-344 (F-344) and Sprague-Dawley (SD) rat hepatocytes were incubated with 1,2-DCB (3.6-12.4 mumol) and examined for enhanced DNA-binding activity of the oxidant-sensitive transcription factors activator protein-1 (AP-1), nuclear factor-kappa B (NF-kappaB), and electrophile responsive element (EpRE), and production and release of the chemokine cytokine-induced neutrophil chemoattractant (CINC). In F-344 rat hepatocytes, the activities of AP-1 and NF-kappaB were increased by as much as 3-fold by 6 h of 1,2-DCB treatment, when compared to control. Nuclear translocation of EpRE was also enhanced by 3-fold and occurred 2 h following 1,2-DCB treatment. These events were greater in F-344 than in SD rat hepatocytes incubated with 1,2-DCB. Moreover, F-344 rat hepatocytes produced and released CINC following incubation with 1,2-DCB, but SD rat hepatocytes did not. Lastly, conditioned media from 1,2-DCB-treated F-344 rat hepatocytes stimulated KC activity as determined by enhanced NF-kappaB-binding activity and increased nitric oxide production. Collectively, these data suggest that the mechanisms of 1,2-DCB-induced hepatotoxicity involve intercellular communication whereby compromised hepatocytes may signal KC activation via the production and release of oxidant-sensitive chemokines and cytokines.
 
Article
The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events.
 
Article
Trichloroethylene (Tri) caused modest cytotoxicity in freshly isolated human proximal tubular (hPT) cells, as assessed by significant decreases in lactate dehydrogenase (LDH) activity after 1 h of exposure to 500 microM Tri. Oxidative metabolism of Tri by cytochrome P-450 to form chloral hydrate (CH) was only detectable in kidney microsomes from one patient out of four tested and was not detected in hPT cells. In contrast, GSH conjugation of Tri was detected in cells from every patient tested. The kinetics of Tri metabolism to its GSH conjugate S-(1,2-dichlorovinyl)glutathione (DCVG) followed biphasic kinetics, with apparent Km and Vmax values of 0.51 and 24.9 mM and 0.10 and 1.0 nmol/min per mg protein, respectively. S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate metabolite of Tri that is considered the penultimate nephrotoxic species, caused both time- and concentration-dependent increases in LDH release in freshly isolated hPT cells. Preincubation of hPT cells with 0.1 mM aminooxyacetic acid did not protect hPT cells from DCVC-induced cellular injury, suggesting that another enzyme besides the cysteine conjugate beta-lyase may be important in DCVC bioactivation. This study is the first to measure the cytotoxicity and metabolism of Tri and DCVC in freshly isolated cells from the human kidney. These data indicate that the pathway involved in the cytotoxicity and metabolism of Tri in hPT cells is the GSH conjugation pathway and that the cytochrome P-450-dependent pathway has little direct role in renal Tri metabolism in humans.
 
Article
Agents that disrupt functions of the endoplasmic reticulum (ER) induce expression of ER stress-response genes including ER chaperones. Increased expression of the major ER chaperone, Grp78, protects cells, including renal epithelial cells, from chemically induced injury and death in vitro. In this study, we determined if pharmacological manipulation of the ER stress-response gene is an effective strategy to protect the kidney from chemical stress in vivo. Treatment with trans-4,5-dihydroxy-1,2-dithiane (DTTox), a novel inducer of ER stress proteins, stimulated a time-and dose-dependent increase in Grp78 expression in the kidney, but it did not cause detectable injury. Furthermore, prior treatment with DTTox protected the proximal tubular epithelium against a subsequent challenge with the nephrotoxicant S-(1,1,2,2,-tetrafluoroethyl)-L-cysteine (TFEC). In contrast, activating a heat shock response did not have a protective effect. Prior treatment with DTTox did not reduce covalent binding of radiolabeled reactive metabolites of (35)S-TFEC to renal proteins, indicating that protection was not due to an effect on the metabolic activation of TFEC to the reactive metabolite(s) responsible for renal injury. Antisense grp78 expression in the renal epithelial cell line LLC-PK1 blocked the DTTox-induced Grp78 increase and ablated the protective effect against TFEC damage, indicating that the induction of grp78 expression and the ER stress response were critical for the protective effect of DTTox. These findings suggest that increased expression of Grp78 plays a major role in the protection of renal epithelial cells from reactive intermediate-induced chemical injury in vivo and that pharmacological manipulation is an effective strategy to prevent damage by some classes of nephrotoxicants.
 
Article
1,3-Butadiene (BD) is an important industrial chemical that is classified as a human carcinogen. BD carcinogenicity has been attributed to its metabolism to several reactive epoxide metabolites and formation of the highly mutagenic 1,2:3,4-diepoxybutane (DEB) has been hypothesized to drive mutagenesis and carcinogenesis at exposures experienced in humans. We report herein the formation of DEB-specific N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) in BD-exposed workers as a biomarker of DEB formation. pyr-Val was determined in BD monomer and polymer plant workers that had been previously analyzed for several other biomarkers of exposure and effect. pyr-Val was detected in 68 of 81 (84%) samples ranging from 0.08 to 0.86 pmol/g globin. Surprisingly, pyr-Val was observed in 19 of 23 administrative control subjects not known to be exposed to BD, suggesting exposure from environmental sources of BD. The mean ± SD amounts of pyr-Val were 0.11 ± 0.07, 0.16 ± 0.12, and 0.29 ± 0.20 pmol/g globin in the controls, monomer, and polymer workers, respectively, clearly demonstrating formation of DEB in humans. The amounts of pyr-Val found in this study suggest that humans are much less efficient in the formation of DEB than mice or rats at similar exposures. Formation of pyr-Val was more than 50-fold lower than has been associated with increased mutagenesis in rodents. The results further suggest that formation of DEB relative to other epoxides is significantly different in the highest exposed polymer workers compared with controls and BD monomer workers. Whether this is due to saturation of metabolic formation or increased GST-mediated detoxification could not be determined.
 
Representative 2D-DIGE patterns of lumbar spinal cords from animals treated with 20 mg/kg/day 1,2-DAB versus 1,3-DAB, 5 days a week, for 2 weeks. The 22 differentially expressed protein spots (circles 1-22) that were differentially modulated by 1,2-DAB versus vehicle (left) and 1,2-DAB versus 1,3DAB (right) were subjected to MALDI-TOF/MS-MS for protein ID.
Article
Motor neuron axonopathy in diseases such as amyotrophic lateral sclerosis can be modeled and probed with neurotoxic chemicals that induce similar patterns of pathology, such as axonal spheroids that represent focal accumulation of anterogradely transported neurofilaments (NFs). The aromatic gamma-diketone-like 1,2-diacetylbenzene (1,2-DAB), but not its 1,3-DAB isomer, reacts with epsilon-amino- or sulfyhydryl groups of (neuro)proteins, forms adducts, and causes NFs to accumulate at proximal sites of elongate motor axons. We exploit the protein-reactive properties of neurotoxic 1,2-DAB versus the nonprotein-reactive properties of non-neurotoxic 1,3-DAB to unveil proteomic changes associated with this type of pathology. We used two-dimensional differential in-gel electrophoresis (2D-DIGE), matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry to analyze the lumbosacral spinal cord proteome of adult Sprague-Dawley rats treated systemically with 20 mg/kg/day 1,2-DAB, equimolar dose of 1,3-DAB, or equivalent volume of vehicle (saline containing 2% acetone), 5 days a week, for 2 weeks. 1,2-DAB significantly altered the expression of protein disulfide isomerase, an enzyme involved in protein folding, and gelsolin, an actin-capping and -severing protein. Modifications of these two proteins have been incriminated in the pathogenesis of nerve fiber degeneration. Protein-reactive and neurotoxic 1,2-DAB appears to be excellent tool to dissect mechanisms of nerve fiber (axon) degeneration.
 
Article
Eight different doses (2.5 to 10.0 mg/kg) of 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin (HpCDD) were administered acutely to a total of 272 female Sprague-Dawley rats. The doses ranged from a NOAEL for wasting/hemorrhage to supralethal doses. Dose- and time-responses of wasting/hemorrhage, anemia, and cancer were and are being studied as end points of toxicity. The experiments will be continued until the last rat dies. There was a very steep dose- and time-response between the LOAEL for wasting/hemorrhage (2.8 mg/kg) and the third highest dose (4.1 mg/kg) of HpCDD. The dose-and time-responses were nearly symmetrical, obeying Haber's Rule of inhalation toxicology (c x t = constant) even beyond 100% mortality. Introduction of a minimum of 25% body weight loss as a discriminatory criterion to separate wasting from hemorrhage as the primary cause of death reduced variability from 5.8 to 3.2%. An arithmetic plot of the dose and time data resulted in a nearly perfect hyperbola. A logarithmic plot of these data yielded a straight line of similar perfection. Dose-response data at constant times illustrate the shifting of the dose-response curve towards a liminal value, which represents the necessary observation period for this effect. Time-response data at constant doses demonstrate the shifting of the time-response curve towards a liminal value, which represents the LOAEL for the dose-response of this effect. A three-dimensional plot of dose- and time-response data depicts the surface area on which c x t is constant along hyperbolas, in terms of wasting as the end point of toxicity. Surviving rats in all groups started developing anemia 126 days after dosing, but no rat died of wasting/hemorrhage after day 74. Rats surviving anemia began to die of lung cancer as of day 397 after dosing. Thus, although the experiment has been completed as far as dose- and time-responses of wasting/hemorrhage are concerned, it will be about another 2 years before complete dose and time responses will become available for anemia and lung cancer.
 
Article
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.
 
Article
Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.
 
Article
Previously, we have shown that exposure of cultured rat cerebellar granule cells to the hydrocarbon solvent 1,2,4-trimethylcyclohexane leads to formation of reactive oxygen species (ROS). However, the cellular mechanisms responsible for formation of ROS in these cells after exposure to organic solvents are poorly understood. Here, we found that 1,2,4-trimethylcyclohexane induced a time and concentration dependent dephosphorylation of Akt-1 at Ser-473 and Raf-1 at Ser-259. An increased level of phosphorylated extracellular signal-regulated kinases (Erk1/2) at Tyr-204 was observed. By use of the nitric oxide synthase inhibitors N(omega)-nitro-L-arginine methylester and diphenyleneiodonium, we found that intracellular formation of nitric oxide was necessary for phosphorylation of Erk1/2 and for the formation of ROS. Furthermore, the ROS formation was inhibited by the Erk1/2 pathway inhibitor U0126. A 1,2,4-trimethylcyclohexane (TMCH)-induced cell death was lowered by U0126 and the free radical scavenger vitamin E. Our results show that Erk1/2 kinases and nitric oxide (NO) may participate in ROS formation induced by 1,2,4-trimethylcyclohexane in cultured rat cerebellar granule cells, and also indicate a crosstalk between Akt and the Raf-Mek-Erk signaling systems.
 
Article
A cluster of occupational asthma (OA) cases associated with occupational exposure to 3-amino-5-mercapto-1,2,4-triazole (AMT) and N-(2,6-difluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide (DE498) in a herbicide producing plant was previously reported by the National Institute for Occupational Safety and Health. Due to the limited toxicological data available for these chemicals, murine studies were undertaken to evaluate the toxicity and sensitization potential of these two agents. No signs of systemic toxicity as evaluated by body and selected organ weights or irritancy were observed following dermal exposure to concentrations up to 25% (w/v) AMT in BALB/c mice. DE498 tested negative for sensitization potential in both the TOPKAT QSAR model and in vivo in the Local Lymph Node Assay (LLNA), while AMT tested positive in both TOPKAT QSAR and the LLNA. Evaluation of the potential for AMT to induce contact hypersensitivity using the MEST yielded negative results. Cytokine evaluation and phenotypic analysis of draining lymph node (DLN) cells demonstrated an increase in IL-4 and IgE+B220+ cells 4 and 10 days post initial exposure, respectively. Following dermal exposure 7 days a week for 35 days, animals exposed to up to 25% AMT demonstrated a dose-dependent elevation in total serum IgE and an increase in airway hyperreactivity upon methacholine challenge. Following intratracheal challenge with AMT, pulmonary histopathology revealed a dose-dependent suppurative and histiocytic alveolitis in these animals. These studies indicate that DE498 does not induce sensitization following dermal exposure; however, AMT was identified as a sensitizer with the potential to induce airway hyperreactivity.
 
Article
Of the twelve different chlorobenzene isomers, a thorough evaluation of carcinogenicity has only been assessed on monochlorobenzene, 1,2-, and 1,4-dichlorobenzene, and hexachlorobenzene. In the studies presented here, we measured the ability of 1,4-dichlorobenzene (DCB), 1,2,4,5-tetrachlorobenzene (TeCB), pentachlorobenzene (PeCB), and hexachlorobenzene (HCB) to promote glutathione S-transferase pi (GSTP1-1) positive preneoplastic foci formation in rat liver, following diethylnitrosamine (DEN) initiation. The results from these studies show that TeCB, PeCB, and HCB all promote the formation of GSTP1-1 positive foci and that DCB does not. The numbers and area of foci were greatest following HCB promotion, and TeCB and PeCB were approximately equal in their promoting ability. Levels of hepatic CYP1A2, CYP2B1/2, non-focal GSTP1-1, and c-fos were measured in response to treatment with the 4 chlorobenzene isomers, as were reduced glutathione (GSH) and oxidized glutathione (GSSG) levels. Results from these studies show that induction of CYP1A2 and CYP2B1/2 have correlation with both the presence and degree of GSTP1-1 foci promotion by the 4 chlorobenzenes. Alterations in GSH and GSSG levels were similar in PeCB- and TeCB-treated animals in that GSSG levels were significantly decreased, whereas HCB and DCB did not have this effect, although HCB treatment led to a significant increase in GSH levels. We conclude that induction of CYP1A2 or CYP2B1/2 by chlorobenzene isomers may indicate promotional ability, and that this property might be exploited to predict the hepatocarcinogenicity of other chlorobenzene isomers.
 
Number of Litters and Number of Pups per Litter 
Mean Pup Weights until Weaning 
PCB Concentrations in Adipose Tissue 
PCB Concentrations in the Brain 
Article
Previous studies revealed effects of polychlorinated biphenyls (PCBs) and other polyhalogenated hydrocarbons on steroid hormone levels and hormone-dependent functions including behavior. In the present study serum concentrations of the vitamin D(3) metabolites 25-hydroxycholecalciferol (25-D) and 1,25-dihydroxycholecalciferol (1,25-D) were determined in rat dams and offspring after exposure to a PCB mixture that was reconstituted according to the congener pattern found in human breast milk. Unmated females were exposed to diets adulterated with 0; 5; 20; or 40 mg PCBs/kg diet. Exposure started 50 days prior to mating and was terminated at birth. Gestational exposure reduced serum concentrations of 1,25-D in dams in a dose-dependent manner. Concentration of 25-D was also decreased at the time of delivery, but not at weaning. Determination of 1,25-D in offspring at weaning revealed reductions in both high-exposure groups. Levels of 25-D were diminished only at the highest exposure level. Internal PCB concentrations in adipose tissue and brains exhibited a linear relation to dosages in diet. Concentrations of PCBs in brains were similar in dams and offspring at birth, but decreased at the end of lactation in dams. In offspring, values increased during this period because of continued exposure via the milk. In the adipose tissue, PCB levels were much lower in offspring than in dams. To our knowledge, this is the first report of PCB-induced effects on vitamin D(3) metabolites. In dams, reductions were seen even at the lowest exposure level used. Further studies are needed to evaluate the biological significance of these reductions in pregnant dams and possible consequences for the developing offspring.
 
Article
Felbamate, 2-phenyl-1,3-propanediol dicarbamate, is a novel anticonvulsant that is effective against both chemically and electrically induced seizures in laboratory animals, Oncogenic studies were conducted in mice and rats to establish a preclinical safety profile for this drug. There was an increased incidence of hepatic cell adenoma in male and female mice and in female rats. There was an increased incidence of interstitial cell tumors of the testes in the male rat. (C) 1998 Society of Toxicology.
 
Article
1,3-Dichloropropene (1,3-D) is a soil fumigant used primarily for pre-planting control of parasitic nematodes. In a previous chronic dietary exposure study, 1,3-D induced an increased incidence of hepatocellular adenomas in male rats at a dose of 25 mg/kg/day . While the mechanism for tumor induction in the rat liver by 1,3-D has not been specifically elucidated, available data suggested that the observed liver tumorigenesis was through a non-genotoxic mode of action at the tumor promotion stage. Fischer 344 rats containing preneoplastic lesions were treated (via gavage) with 25 mg/kg/day 1,3-D or 80 mg/kg/day phenobarbital (PB) for 30 days and 60 days, or for 30 days followed by a 30-day recovery period (no compound exposure). Following treatment, placental form GST (GSTP) positive and GSTP-negative liver focal lesions were quantitated as to size and number. 1,3-D treatment had no effect on GSTP-positive foci number or relative size but significantly increased the number, labelling index , and relative size of GSTP-negative focal lesions (identified by H and E staining) after 30 and 60 days of treatment. Following the 30-day recovery period, the number, labelling index and relative size of the GSTP-negative lesions in 1,3-D treated animals returned to control levels. As expected, Phenobarbital treatment produced an increase in number and relative size of the GSTP-positive lesions. The results of this study are consistent with 1,3-D inducing liver carcinogenesis through a non-genotoxic mode-of action by functioning as a tumor promoter specifically through induction of a non-GSTP staining focal hepatocyte population.
 
Article
The potential oral toxicity of 1,3-dichloropropene (1,3-D) has been evaluated in a number of dietary toxicity studies. The relatively high vapor pressure of 1,3-D, its short half-life in drinking water, and its reactivity with constituents of feed necessitated the use of a microencapsulated formulation (starch-sucrose shell) of 1,3-D in these studies. The bioavailability of ingested microencapsulated 1,3-D was determined by characterizing and comparing the kinetics of 1,3-D in the blood of female F344 rats coadminis-tered microencapsulated 1,3-D and neat 13C-1,3-D (25 mg/kg each) via gavage. Blood concentrations of total or cis- and trans-is-isomers of 1,3-D in treated rats were determined using gas chromatography-mass spectroscopy (GC-MS) or in situ membrane extraction MS. Urine was also collected and analyzed by GC-MS for the presence of the mercapturate excretion product of 1,3-D [N-acetyl-S-(3-chloropropenyl-2)L-cysteine; 1,3-DMA]. Blood levels of 1,3-D and 13C-1,3-D displayed similar kinetics, peaking within 10 min of dosing followed by a rapid biphasic elimination. Higher peak blood levels and greater blood curve areas (AUC) were attained for trans- than cis-l,3-D and 13C-1,3-D and greater amounts of cis- than trans-l,3-DMA and 13C-1,3-DMA were excreted in the urine consistent with the known rapid and disproportionate glutathione conjugation of the cis-isomer in the gastric mucosa. Slightly higher cis-l,3-D than cis-13C-l,3-D blood levels and AUCs were also consistently noted while the reverse was true for urinary excretion of cis-13C-13-DMA and cis-l,3-DMA suggesting that 1,3-D derived from microencapsulated test material may be absorbed and/or metabolized in the stomach mucosa at a slightly slower rate than that from neat material. The latter, however, would be of no consequence during the administration of 1,3-D to animals via their diets as competing test materials would not be present and 1,3-D blood kinetics were unaffected. Overall, the results of this study demonstrate the ready bioavailability of microencapsulated 1,3-D and rapid elimination of 1,3-D from the blood of rats.
 
Cytotoxic responses of C6 glioma cells exposed to increasing concentrations of 1,3-DNB (50lM to 1mM) for 36 h with or without the addition of ALCAR (5mM). (A) Cellular reducing potential was determined using the MTT reduction assay. Data are expressed as percent MTT reduction compared with vehicle control (DMSO). (B) Quantification of released LDH was determined using the INT-coupled enzymatic assay. Data are expressed as mean ± SEM (n ¼ 4). Significant differences from vehicle control (DMSO) are denoted as follows: *p < 0.05, **p < 0.01. (C) Representative photomicrographs depicting 1,3-DNB (500lM and 1mM)-induced morphological changes after 36-h exposure in the presence or absence of ALCAR (5mM).
Modification of LA by exposure to 1,3-DNB in C6 glioma cells. (A) Representative immunoblot illustrating the dose-dependent effects on LA immunoreactivity by 36-h exposure to 1,3-DNB and (B) time-dependent effects on LA immunoreactivity by exposure to 500lM 1,3-DNB for 6, 12, 24, and 48 h. Quantification of (C) dose-dependent and (D) time-dependent LA immunoreactivity loss by densitometric analysis. (E) Correlation analysis between loss of LA immunoreactivity and PDHc activity. Data are presented as mean pixel intensity of LA normalized to complex II expression. Significant differences from control are denoted as follows: *p < 0.05.
Article
Prolonged exposure to the chemical intermediate, 1,3-dinitrobenzene (1,3-DNB), produces neuropathology in the central nervous system of rodents analogous to that observed in various conditions of acute energy deprivation including thiamine deficiency and Leigh's necrotizing encephalopathy. Increased production of reactive intermediates in addition to induction of oxidative stress has been implicated in the neurotoxic mechanism of 1,3-DNB, but a clear metabolic target has not been determined. Here we propose that similar to thiamine deficiency, the effects of 1,3-DNB on metabolic status may be due to inhibition of the thiamine-dependent α-ketoacid dehydrogenase complexes. The effects of 1,3-DNB on astroglial metabolic status and α-ketoacid dehydrogenase activity were evaluated using rat C6 glioma cells. Exposure to 1,3-DNB resulted in altered morphology and biochemical dysfunction consistent with disruption of oxidative energy metabolism. Cotreatment with acetyl-carnitine or acetoacetate attenuated morphological and metabolic effects of 1,3-DNB exposure as well as increased cell viability. 1,3-DNB exposure inhibited pyruvate dehydrogenase complex (PDHc) and the inhibition correlated with the loss of lipoic acid (LA) immunoreactivity, suggesting that modification of LA is a potential mechanism of inhibition. Treatment with antioxidants and thiol-containing compounds failed to protect against loss of LA. Alternatively, inhibition of dihydrolipoamide dehydrogenase, the E3 component of the complex attenuated loss of LA. Collectively, these data suggest that 1,3-DNB impairs oxidative energy metabolism through direct inhibition of the PDHc and that this impairment is due to perturbations in the function of protein-bound LA.
 
Investigated metabolic pathways of BD and its metabolites. For simplification, the optical isomers of the BD metabolites are not shown. 
System for once-through perfusions of volatile or gaseous substances through rat or mouse livers (for detailed description see text). a, magnetic stirrer; b, perfusate reservoir; c, peristaltic pump; d, oxygenator; e, gas bubble trap; f, closed all-glass rat liver chamber; g, closed all-glass mouse liver chamber; h, thermometer; i, septum for collecting influent perfusate samples; j, septum for collecting effluent perfusate samples; k, all-glass sphere (64 l); l, magnetic stirring motor and magnetic propeller; m, vacuum gauge; n, membrane pump; o, Teflon piston pump; p, silicon tube containing flowing water (37°C). 
Typical concentration-time courses of BD and its metabolites EB, B-diol, and DEB in influent and effluent perfusate of a BD-perfused mouse (A) and rat (B) liver. Symbols: measured compound concentrations in influent (hollow) and effluent (filled) perfusate; dashed lines: mean concentrations 
Article
The industrial chemical 1,3-butadiene (BD) is a potent carcinogen in mice and a weak one in rats. This difference is generally related to species-specific burdens by the metabolites 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD), which are all formed in the liver. Only limited data exist on BD metabolism in the rodent liver. Therefore, metabolism of BD, its epoxides, and the intermediate 3-butene-1,2-diol (B-diol) was studied in once-through perfused livers of male B6C3F1 mice and Sprague-Dawley rats. In BD perfusions, predominantly EB and B-diol were found (both species). DEB and EBD were additionally detected in mouse livers. Metabolism of BD showed saturation kinetics (both species). In EB perfusions, B-diol, EBD, and DEB were formed with B-diol being the major metabolite. Net formation of DEB was larger in mouse than in rat livers. In both species, hepatic clearance (ClH) of EB was slightly smaller than the perfusion flow. In DEB perfusions, EBD was formed as a major metabolite. ClH of DEB was 61% (mouse) and 73% (rat) of the perfusion flow. In the B-diol-perfused rat liver, EBD was formed as a minor metabolite. ClH of B-diol was 53% (mouse) and 34% (rat) of the perfusion flow. In EBD-perfused rat livers, ClH of EBD represented only 22% of the perfusion flow. There is evidence for qualitative species differences with regard to the enzymes involved in BD metabolism. The first quantitative findings in whole livers showing intrahepatic first-pass metabolism of BD and EB metabolites will improve the risk estimation of BD. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] /* */
 
Article
1,3-Butadiene (butadiene) is a potent carcinogen in mice, but not in rats. Metabolic studies may provide an explanation of these species differences and their relevance to humans. Male Sprague-Dawley rats and B6C3F1 mice were exposed for 6 h to 200 ppm [2,3-14C]-butadiene (specific radioactivity [sa] 20 mCi/mmol) in a Cannon nose-only system. Radioactivity in urine, feces, exhaled volatiles and 14C-CO2 were measured during and up to 42 h after exposure. The total uptake of butadiene by rats and mice under these experimental conditions was 0.19 and 0.38 mmol (equivalent to 3.8 and 7.5 mCi) per kg body weight, respectively. In the rat, 40% of the recovered radioactivity was exhaled as 14C-CO2, 70% of which was trapped during the 6-h exposure period. In contrast, only 6% was exhaled as 14C-CO2 by mice, 3% during the 6-h exposure and 97% in the 42 h following cessation of exposure. The formation of 14C-CO2 from [2,3-14C]-labeled butadiene indicated a ready biodegradability of butadiene. Radioactivity excreted in urine accounted for 42% of the recovered radioactivity from rats and 71% from mice. Small amounts of radioactivity were recovered in feces, exhaled volatiles and carcasses. Although there was a large measure of commonality, the exposure to butadiene also led to the formation of different metabolites in rats and mice. These metabolites were not found after administration of [4-14C]-1,2-epoxy-3-butene to animals by i.p. injection. The results show that the species differences in the metabolism of butadiene are not simply confined to the quantitative formation of epoxides, but also reflect a species-dependent selection of metabolic pathways. No metabolites other than those formed via an epoxide intermediate were identified in the urine of rats or mice after exposure to 14C-butadiene. These findings may have relevance for the prediction of butadiene toxicity and provide a basis for a revision of the existing physiologically based pharmacokinetic models.
 
Article
Felbamate, 2-phenyl-1,3-propanediol dicarbamate, is a novel anticonvulsant that is effective against both chemically and electrically induced seizures in laboratory animals. Acute, subchronic, and chronic studies were conducted in mice, rats, and dogs to establish a preclinical safety profile for this drug. Clinical signs following single intraperitoneal doses included hypoactivity, tremors, decreased muscle tone, ataxia, prostration, and labored breathing. Death was observed after intraperitoneal but not oral administration. A consistent drug-related effect noted in all multiple-dose studies with this compound was decreased body weight and food consumption. The only other consistent change noted in multiple-dose studies with felbamate was an increase in liver weight (relative and absolute) in the rat and dog which was accompanied in some cases by increases in serum enzyme levels. No histopathological changes were observed in the liver that could explain these elevated serum enzyme levels. Based on the results of these studies it was concluded that long-term administration of felbamate in human clinical trials was warranted.
 
Article
Regions of the brain with high energy requirements are especially sensitive to perturbations in mitochondrial function. Hence, neurotoxicant exposures that target mitochondria in regions of high energy demand have the potential to accelerate mitochondrial damage inherently occurring during the aging process. 1,3-Dinitrobenzene (DNB) is a model neurotoxicant that selectively targets mitochondria in brainstem nuclei innervated by the eighth cranial nerve. This study investigates the role of age in the regional susceptibility of brain mitochondria-related proteins (MRPs) to oxidation following exposure to DNB. Male F344 rats (1 month old [young], 3 months old [adult], 18 months old [aged]) were exposed to 10 mg/kg DNB prior to mitochondrial isolation and histopathology experiments. Using a high-throughput proteomic approach, 3 important region- and age-related increases in DNB-induced MRP oxidation were determined: (1) brainstem mitochondria are ×3 more sensitive to DNB-induced oxidation than cortical mitochondria; (2) oxidation of brainstem MRPs is significantly higher than in cortical counterparts; and (3) MRPs from the brainstems of older rats are significantly more oxidized than those from young or adult rats. Furthermore, lower levels of DNB cause signs of intoxication (ataxia, chromodacryorrhea) and vacuolation of the susceptible neuropil in aged animals, while neither is observed in DNB-exposed young rats. Additionally, methemoglobin levels increase significantly in DNB-exposed adult and aged animals, but not young DNB-exposed animals. This suggests that oxidation of key MRPs observed in brainstem of aged animals is necessary for DNB-induced signs of intoxication and lesion formation. These results provide compelling evidence that environmental chemicals such as DNB may aid in the acceleration of injury to specific brain regions by inducing oxidation of sensitive mitochondrial proteins.
 
Article
Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC(50) of 284 μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01-0.05%, wt/vol), Nonidet P-40 (0.0015-0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2 nM) did not substantially alter the 1,3-DNB IC(50) value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149-1043 μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (K(I) = 520 ± 100 μM, n = 1 ± 0.6) and the ADA-adenosine complex (K(IS) = 262 ± 7 μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10-500 μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation.
 
Article
Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(1-chloropropyl) phosphate (TCPP) belong to a group of chemicals collectively known as triester organophosphate flame retardants (OPFRs). OPFRs are used in a wide range of consumer products and have been detected in biota, including free-living avian species; however, data on toxicological and molecular effects of exposure are limited. An in vitro screening approach was used to compare concentration-dependent effects of TDCPP and TCPP on cytotoxicity and messenger RNA (mRNA) expression in cultured hepatocytes and neuronal cells derived from embryonic chickens. TDCPP was toxic to hepatocytes (LC₅₀ = 60.3 ± 45.8μM) and neuronal cells (LC₅₀ = 28.7 ± 19.1μM), whereas TCPP did not affect viability in either cell type up to the highest concentration administered, 300μM. Real-time reverse transcription-PCR revealed alterations in mRNA abundance of genes associated with phase I and II metabolism, the thyroid hormone (TH) pathway, lipid regulation, and growth in hepatocytes. None of the transcripts measured in neuronal cells (D2, D3, RC3, and Oct-1) varied in response to TDCPP or TCPP exposure. Exposure to ≥ 10μM TDCPP and TCPP resulted in significant upregulation of CYP2H1 (4- to 8-fold), CYP3A37 (13- to 127-fold), and UGT1A9 (3.5- to 7-fold) mRNA levels. Transthyretin was significantly downregulated more than twofold by TCPP at 100μM; however, TDCPP did not alter its expression. Liver fatty acid-binding protein, TH-responsive spot 14-α, and insulin-like growth factor-1 were all downregulated (up to 10-fold) in hepatocytes exposed to ≥ 0.01μM TDCPP and TCPP. Taken together, our results indicate that genes associated with xenobiotic metabolism, the TH pathway, lipid regulation, and growth are vulnerable to TDCPP and TCPP administration in cultured avian hepatocytes. The mRNA expression data were similar to those from a previous study with hexabromocyclododecane.
 
Polymorphic sites, allelic frequency, and genotype frequency in the 5#-flanking region of the mEH gene among butadiene-exposed workers a 
(A) Estimated change in VFs from background (baseline) level a , computed using the general linear purely additive model, as it relates to BD exposure (Exp) alone, polymorphisms in g1, g2, and g3 alone (corresponding to the presence of the homozygous or heterozygous variant alleles in the ÿ200 linkage, ÿ600 linkage, and the ÿ399 site, respectively). (B) Estimated change in variant frequencies (VFs) from background (baseline) level a , computed using the 4-way firstorder interaction model, as it relates to the interaction between exposure (Exp) and the presence of the polymorphisms in g1, g2, and g3 (see A, above). ''Exp g'' is the interaction term for exposure and polymorphisms at one site at a time, when the other two linkage sites are at their control level (homozygous wild-type). * Statistically significant (p value corrected for the number of comparisons ¼ 0.0027). a The background (baseline) VF, defined as the VF (3 10 ÿ6 ) ± S.E.M. at low levels of BD exposure (<150 ppb), where all the genotypes under study are at their control levels (homozygous wild-type), was estimated to be 4.02 ± 1.32. 
Article
The carcinogenic effects of 1,3-butadiene (BD), a mutagenic chemical widely used in the manufacture of synthetic rubber, are likely initiated through its epoxide metabolites. In humans, these epoxides are detoxified predominantly by hydrolysis, a reaction mediated by the microsomal epoxide hydrolase (mEH; EPHX1) enzyme. It appears reasonable to hypothesize that BD-exposed individuals possessing lower mEH detoxification capacity may have elevated risk of adverse health effects. The interindividual levels of mEH enzymatic activity vary considerably, and polymorphisms in the mEH gene may contribute to this variability. In addition to the well-studied coding region polymorphisms encoding Tyr113His and His139Arg substitutions, seven other polymorphic sites in the 5'-flanking region of the mEH gene have been reported. These polymorphisms appear to differentially affect mEH gene transcriptional activities. The 5'-flanking region polymorphisms exist in two linkages, the -200 linkage (-200C/T, -259C/T, -290T/G) and the -600 linkage (-362A/G, -613T/C, -699T/C), whereas the -399T/C polymorphism exists as an independent site. Because these polymorphisms may affect total mEH enzymatic activity, we hypothesized that they influence the mutagenic response associated with occupational exposure to BD. We genotyped the 5'-region of the mEH gene in 49 non-smoking workers from two styrene-butadiene rubber facilities in southeast Texas and evaluated the linkage patterns against results obtained from an autoradiographic HPRT mutant lymphocyte assay, used as a biomarker of genotoxic effect. In the study population, 67% were exposed to low BD levels, <150 parts per billion, and 33% were exposed to >150 ppb. We used the observed HPRT mutant (variant) frequency (VF) in the studied population and a 4-way first-order interaction statistical model to estimate parameters that describe the influence of exposure, genotypes and the interaction between the two on the HPRT VF in the target population. The background (baseline) VF, defined as the VF (x 10(-6)) +/- S.E.M. at low levels of BD exposure (<150 ppb) where all the genotypes under study are homozygous wild-type, was estimated to be 4.02 +/- 1.32. Exposure to >150 ppb of BD alone resulted in an estimated increase in VF of 3.42 +/- 2.47 above the baseline level. Inheritance of the variant ATT allele in the -600 linkages resulted in an estimated increase in VF of 3.39 +/- 1.67 above the baseline level. When the interaction between BD exposure and the ATT allele in the -600 linkage group was considered, a statistically significant positive interaction was observed, with an estimated increase in the VF of 10.89 +/- 2.16 (95% CI = 6.56-15.20; p = 0.0027) above baseline. These new data confirm and extend our previous findings that sensitivity to the genotoxic effects of BD is inversely correlated with predicted mEH activity.
 
Article
1,3-Butadiene (BD), a compound used extensively in the rubber industry, is weakly carcinogenic in Sprague-Dawley rats after chronic exposures to concentrations of 1000 and 8000 ppm. Conversely, in B6C3F1 mice, tumors occur after chronic exposures to concentrations as low as 6.25 ppm. Previously, we have shown that tissue concentrations of the mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), are present in greater concentrations in mice than in rats following acute exposures to low levels (100 ppm or less). This disparity was particularly significant for the diepoxide. We hypothesized that if these epoxides are involved in the carcinogenic response of BD, then they will also be present in rat tissues at relatively high concentrations following exposures to 8000 ppm BD. In the present study, concentrations of the BD epoxides, BDO and BDO2, were determined in blood of female Sprague-Dawley rats following a single 6-h exposure and 10 repeated exposures to a target concentration of 8000 ppm BD. Concentrations of these epoxides were also determined in a number of other tissues, including the primary rat target organ-mammary gland-following 10 repeated exposures. Blood concentrations of BDO were 4030 pmol/g +/- 191 following a 6-h exposure and were 18% lower following the 10-day exposure. Blood concentrations of BDO2, following the 8000 ppm exposures, were very similar to those previously observed after exposures to 62.5 ppm BD (11 +/- 1 and 17 +/- 1 pmol/g following exposures of 6h and 6h/day for 10 days, respectively.) Concentrations of BDO ranged from 740 +/- 110 (femur) to 8990 +/- 1150 (fat) pmol/g tissue. Concentrations of BDO2 were similar among eight tissues analyzed, ranging from 5 +/- 1 (femur) to 17 +/- 3 (heart) pmol/g tissue. Tissue concentrations of butadiene monoepoxide were increased by 17- to 50-fold in tissues from rats exposed by inhalation to 8000 ppm BD as compared to tissues from rats exposed to 62.5 ppm BD. Based on earlier studies at our institute the internal dose of BD increases approximately 14-fold in the 8000 ppm-exposed rats compared to rats exposed to 62.5 ppm BD. Concentrations of butadiene diepoxide in rat tissues following an exposure to 8000 ppm BD were similar to those observed in rat tissues following exposures to 62.5 ppm BD. This study shows that pathways responsible for the accumulation of BDO2 in rats are saturated following low-level BD exposures. This suggests that the primary determinant of BD tumorigenicity in rats is not butadiene diepoxide. The high levels of BDO observed in rat mammary tissue suggest that this metabolite may be a more important determinant of BD carcinogenesis in the rat.
 
Article
1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity.
 
Summary of Most Relevant Dose-Response Studies Evaluated 
Article
1,3-butadiene (BD) is a smoke component selected by the World Health Organization (WHO) study group on Tobacco Product Regulation (TobReg) for mandated lowering. We examined the tobacco-smoke related health effects induced by BD and possible health impacts of risk reduction strategies. BD levels in mainstream smoke (MSS) from international and Canadian cigarettes, and environmental tobacco smoke (ETS) were derived from scientific journals and international government reports. Dose-response (DR) analyses from toxicity studies from government reports were evaluated and the most sensitive cancer and non-cancer endpoints were selected. The risks were evaluated by taking the ratio (margin of exposure, MOE) from the most sensitive toxicity endpoint and appropriate exposure estimates for BD in MSS and ETS. BD is a good choice for lowering given that MSS and ETS were at levels for cancer (leukemia) and non-cancer (ovarian atrophy) risks, and the risks can be significantly lowered when lowering the BD concentrations in smoke. Several risk reduction strategies were analyzed including: a maximum level of 125% of the median BD value per mg nicotine obtained from international brands as recommended by the WHO TobReg, tobacco substitute sheets, dual and triple carbon filters, and polymer-derived carbon. The use of tobacco substitute sheet with a polymer-derived carbon filter resulted in the most significant change in risk for cancer and non-cancer effects. Our results demonstrate that MOE analysis might be a practical way to assess the impact of risk reduction strategies on human health in the future.
 
Article
1,3-Butadiene (BD) is carcinogenic in mice and rats, with mice being considerably more sensitive than rats. Urine metabolites are 1, 2-dihydroxybutyl mercapturic acid (DHBMA) and a mixture of monohydroxy-3-butenyl mercapturic acids (MHBMA). The reactive metabolite 1,2-epoxy-3-butene forms 1- and 2-hydroxy-3-butenyl valine adducts in hemoglobin (MHBVal). The objectives of the study were (1) to compare the suitability of MHBMA, DHBMA, and MHBVal as biomarkers for low levels of exposure to BD, and (2) to explore relative pathways of metabolism of BD in humans for comparison with mice and rats, which is important in relation to cancer risk assessment in man. Analytical methods of measuring MHBMA, DHBMA, and MHBVal were modified and applied in 2 studies to workers engaged in the manufacture and use of BD. Airborne BD concentrations were assessed by personal air monitoring. MHBMA in urine was more sensitive for monitoring recent exposures to BD when compared to DHBMA and could measure 8-h time weighted average exposures as low as 0.13 ppm. Relatively high natural background levels in urine restricted the sensitivity of DHBMA. The origin of this background is currently unknown. The measurement of MHBVal adducts in hemoglobin was a sensitive method for monitoring cumulative exposures to BD at or above 0.35 ppm. Statistically significant relationships were found between urinary MHBMA and DHBMA concentrations, between either of these variables and 8-h airborne BD levels and between MHBVal adducts and average airborne BD levels over 60 days. The data on biomarkers demonstrated a much higher rate of hydrolytic metabolism of 1,2-epoxy-3-butene in humans compared to mice and rats, which was reflected in a much higher DHBMA/(DHBMA + MHBMA) ratio and in much lower levels of MHBVal in humans. Assuming a genotoxic mechanism, the data of this study, coupled with other published data on DNA and hemoglobin binding in mice and rats, suggest that the cancer risk for man from exposure to BD is expected to be less than for the rat and much less than for the mouse.
 
Amounts of THB-Gua-BD adducts in liver DNA from mice exposed to 0- or 625-ppm BD. Data are presented as mean ± SD ( n 1⁄4 5). Asterisk and ampersand (* and &) denote significant ( p < 0.05) differences in the same DNA adduct as compared with the corresponding strain’s control mice or as compared with BD-treated mice of other strains, respectively. 
Effects of BD exposure on the extent of DNA methylation in mouse liver. (A) Loss of global DNA methylation in the livers of BD-exposed mice as measured by a cytosine extension ([ 3 H]dCTP incorporation) DNA methylation assay. (B) Loss of LINE1 repetitive elements methylation in the livers of BD- exposed mice as measured by a methylation-sensitive McrBC-qPCR assay. Data are presented as fold change in BD (625 ppm)-exposed mice relative to the control mice in each strain, mean ± SD ( n 1⁄4 5). Asterisks (*) denote a significant ( p < 0.05) difference from the corresponding strain’s control group. 
Heatmap of differentially expressed hepatotoxicity biomarker genes in the livers of control and BD-exposed C57BL/6J, A/J, and CAST/EiJ mice. Gene expression was determined in total liver RNA from control and BDexposed mice (n ¼ 3 from each group). A heatmap (average of fold change between control and exposed groups within each strain) of genes identified as significant (p < 0.05) and changing in expression by at least 1.5-fold is shown. The color bar identifies up and downregulated genes as indicated. See Supplementary table 1 for a list of genes and gene expression values.
Analysis of chromatin structure in the livers of control and BDexposed C57BL/6J, A/J, and CAST/EiJ mice. (A) Chromatin structure in the livers of control and BD-exposed C57BL/6J, A/J, and CAST/EiJ mice was determined by analyzing the accessibility of CCGG sites within nucleosomal DNA to methylation. The extent of [ 3 H]dCTP incorporation into DNA is directly proportional to changes in chromatin condensation. (B) Total liver RNA from control mice (white bars) and mice exposed to 625-ppm BD (black bars) was used to evaluate transcript abundance of Ezh2. Data are presented as mean ± SD (n ¼ 5). Asterisks (*) denote a significant (p < 0.05) difference from the corresponding strain's control group.
Article
1,3-Butadiene (BD) is a common environmental contaminant classified as "carcinogenic to humans." Formation of BD-induced DNA adducts plays a major role in its carcinogenicity. BD is also an epigenotoxic agent (i.e., it affects DNA and histone methylation in the liver). We used a panel of genetically diverse inbred mice (NOD/LtJ, CAST/EiJ, A/J, WSB/EiJ, PWK/PhJ, C57BL/6J, and 129S1/SvImJ) to assess whether BD-induced genotoxic and epigenotoxic events may be subject to interstrain differences. Mice (male, 7 weeks) were exposed via inhalation to 0 or 625 ppm BD for 6 h/day and 5 days/week for 2 weeks and liver BD-DNA adducts, epigenetic alterations, and liver toxicity were assessed. N-7-(2,3,4-trihydroxybut-1-yl)-guanine adducts were detected in all strains after exposure, yet BD-induced DNA damage in CAST/EiJ mice was two to three times lower. Epigenetic effects of BD were most prominent in C57BL/6J mice where loss of global DNA methylation and loss of trimethylation of histone H3 lysine 9, histone H3 lysine 27, and histone H4 lysine 20, accompanied by dysregulation of liver gene expression indicative of hepatotoxicity, were found. Interestingly, we observed an increase in histone methylation in the absence of changes in gene expression and DNA methylation in CAST/EiJ strain. We hypothesized that mitigated genotoxicity of BD in CAST/EiJ mice may be due to chromatin condensation. Indeed, we show that in response to BD exposure, chromatin condensation occurs in CAST/EiJ, whereas the opposite effect is observed in C57BL/6J mice. These findings demonstrate that interstrain susceptibility to genotoxicity by a well-known environmental carcinogen may be due to strain-specific epigenetic events in response to the exposure.
 
(A) Testis from a rat in the control group; the capsule (left) and the seminiferous tubules are normal with sperm at varying stages of maturation. (B) Testis from a rat in the high dose DMH group. There is complete loss of seminiferous epithelium in a tubule in the upper right with reduced diameter and several others with loss of developing spermatids. There is an intraepithelial vacuole (upper left) and several spermatid giant cells in the tubule in the upper center. The tubule in the upper left has lost all but the germinal layer of cells and those in the lumen have been sloughed from a more proximal area.. Hematoxylin and eosin stain 130.
Article
Pentyl ether (PE) and two newly synthesized polyoxy ethers, 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been proposed as candidate diesel fuel additives. To characterize and compare their toxicity and to provide information for risk assessment, a 4-week oral study was conducted on these compounds. Male Sprague-Dawley rats (288 +/- 20 g) were divided into groups of seven animals each, and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg body weight), or high (200 mg/kg body weight) doses of PE, DEB, or DMH, respectively, 5 days/week for 4 weeks. Animals in the control group received the vehicle (corn oil, 1 ml/100 g body weight) only. At the end of the exposure period, relative testis and thymus weights were reduced by 30 and 46%, respectively, in animals treated with the high dose of DMH. Significant reductions in serum lactate dehydrogenase (LDH), serum uric acid, and blood platelet counts were also observed in the high dose of DMH. Serum corticosterone was significantly depressed in the high doses of PE and DEB and in the low dose of DMH. Serum thiobarbituric acid-reactive substances (TBARS) were decreased (p < 0.05) in all DMH treatment groups and in the medium and high dose PE and DEB groups, while liver TBARS were unaffected by treatment. In the liver, increased glutathione (GSH) level and glutathione-S-transferases activity were detected in the high dose DMH group. Urinary ascorbic acid levels were markedly increased in animals receiving the high doses of PE, DEB, and DMH. Urinary formic acid was increased by 13 times in the high dose PE and DEB groups. Testes of all animals receiving the high dose of DMH showed a moderate to marked degree of degeneration of the seminiferous tubules, including a mild degree of vacuolation. At the same time, the epididymis of these animals had substantially reduced sperm density with prominent presence of spermatid giant cells. Mild histological changes were seen in the liver at all dose levels for all three chemicals. Thyroid effects were also observed in the high dose PE and DEB groups and in the medium and high dose DMH groups. It was concluded that DMH is the most toxic of the three ethers tested, with testicular, epidiymal, and thymic effects being the most prominent at 200 mg/kg. Other significant changes included depressed platelet counts and serum biochemical changes. Increased production of formic acid, an ocular toxin, from PE and DEB treatments may also be of toxicological concern.
 
Top-cited authors
Melvin Andersen
  • ScitoVation
Russell Scott Thomas
  • United States Environmental Protection Agency
Harvey Clewell
Michael S Denison
  • University of California, Davis
Linda S Birnbaum
  • National Institutes of Health